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In routine assays when the linearity of the system has been subject to agreement by the competent authority. However,
demonstrated over an adequate number of experiments
in all cases of dispute, a three-point assay as described above
using a three-point assay, a two-point assay may be sufficient, must be applied.
Table 2.7.2.-1. Diffusion assay
Antibiotic
Solvent to be used
Buffer solution
Reference substance in preparing the
(pH)
stock solution
Micro-organism
Incubation
temperature
Amphotericin B
Amphotericin B CRS
pH 10.5 (0.2 M)
Saccharomyces
cerevisiae
ATCC 9763
IP 1432-83
F - pH 6.1
35-37 C
Bacitracin zinc
0.01 M
Bacitracin zinc CRS
hydrochloric acid
pH 7.0 (0.05 M)
Micrococcus luteus
NCTC 7743
CIP 53.160
ATCC 10240
A - pH 7.0
35-39 C
Bleomycin sulphate
Bleomycin
sulphate CRS
pH 6.8 (0.1 M)
Mycobacterium
smegmatis
ATCC 607
G - pH 7.0
35-37 C
B - pH 7.3
35-39 C
Bacillus subtilis
NCTC 8236
CIP 1.83
A - pH 7.9
30-37 C
Bacillus subtilis
NCTC 10400
CIP 52.62
ATCC 6633
A - pH 7.9
30-37 C
Bacillus subtilis
NCTC 10400
CIP 52.62
ATCC 6633
A - pH 7.9
30-37 C
Bacillus subtilis
NCTC 10400
CIP 52.62
ATCC 6633
E - pH 7.9
30-37 C
Bacillus pumilus
NCTC 8241
CIP 76.18
E - pH 7.9
30-37 C
Bacillus pumilus
NCTC 8241
CIP 76.18
A - pH 7.9
35-39 C
Staphylococcus
epidermidis
NCIB 8853
CIP 68.21
ATCC 12228
A - pH 7.9
35-39 C
pH 5.6
Bacillus subtilis
CIP 52.62
ATCC 6633
NCTC 10400
A - pH 6.6
35-37 C
pH 5.6
Bacillus subtilis
CIP 52.62
ATCC 6633
NCTC 10400
A - pH 6.6
35-37 C
Colistimethate
sodium
Dihydrostreptomycin sulphate
Erythromycin
estolate
Framycetin sulphate
Gentamicin
sulphate
Colistimethate
sodium CRS
Erythromycin CRS
Framycetin
sulphate CRS
Dimethyl
sulphoxide R
Water R
Water R
pH 6.0 (0.05 M)
Bordetella
bronchiseptica
NCTC 8344
CIP 53.157
ATCC 4617
Escherichia coli
NCIB 8879
CIP 54.127
ATCC 10536
Water R
Methanol R (see
the monographs)
Water R
Gentamicin
sulphate CRS
Water R
Josamycin
Josamycin CRS
Methanol R (see
the monograph)
Josamycin
propionate
Josamycin
propionate CRS
Methanol R (see
the monograph)
pH 8.0 (0.05 M)
Bacillus pumilus
NCTC 8241
CIP 76.18
pH 8.0 (0.05 M)
pH 8.0 (0.05 M)
pH 8.0 (0.05 M)
189
Antibiotic
Solvent to be used
Buffer solution
Reference substance in preparing the
(pH)
stock solution
Incubation
temperature
Bacillus subtilis
NCTC 10400
CIP 52.62
ATCC 6633
A - pH 7.9
30-37 C
Staphylococcus
aureus
NCTC 7447
CIP 53.156
ATCC 6538 P
A - pH 7.9
35-39 C
Bacillus pumilus
NCTC 8241
CIP 76.18
E - pH 7.9
30-37 C
Bacillus subtilis
NCTC 10400
CIP 52.62
ATCC 6633
E - pH 7.9
30-37 C
A - pH 7.9
32-35 C
F - pH 6.0
30-37 C
F - pH 6.0
30-32 C
pH 7.0 (0.05 M)
Micrococcus luteus
NCTC 8340
CIP 53.45
ATCC 9341
A - pH 6.6
35-39 C
pH 8.0 (0.05 M)
Bacillus subtilis
NCTC 10400
CIP 52.62
ATCC 6633
A - pH 7.9
30-32 C
Bacillus subtilis
NCTC 8236
CIP 1.83
A - pH 7.9
30-37 C
Bacillus subtilis
NCTC 10400
CIP 52.62
ATCC 6633
A - pH 7.9
30-37 C
Kanamycin
monosulphate
Kanamycin
Water R
monosulphate CRS
pH 8.0 (0.05 M)
Kanamycin acid
sulphate
Neomycin sulphate
Neomycin sulphate for microbiological Water R
assay CRS
Netilmicin
Netilmicin sulphate
sulphate CRS
Water R
pH 8.0 (0.05 M)
Micro-organism
Staphylococcus
aureus
ATCC 6538P
CIP 53.156
pH 8.0 0.1
Candida tropicalis
CIP 1433-83
NCYC 1393
Nystatin
Nystatin CRS
Rifamycin sodium
Rifamycin
sodium CRS
Spiramycin
Streptomycin
sulphate
Spiramycin CRS
Methanol R
Methanol R
pH 6.0 (0.05 M)
containing 5 per
Saccharomyces
cent V/V of dimeth- cerevisiae
ylformamide R
NCYC 87
CIP 1432-83
ATCC 9763
Streptomycin
sulphate CRS
Water R
pH 8.0 (0.05 M)
Tylosin CRS
A mixture of
40 volumes of
methanol R and
60 volumes of 0.1 M
phosphate buffer
solution pH 8.0 R
Micrococcus luteus
NCTC 8340
CIP 53.45
ATCC 9341
A - pH 8.0
32-35 C
Vancomycin
hydrochloride CRS
Water R
pH 8.0
Bacillus subtilis
NCTC 8236
CIP 52.62
ATCC 6633
A - pH 8.0
37-39 C
Tylosin for
veterinary use
Tylosin tartrate for
veterinary use
Vancomycin
hydrochloride
Dimethylformamide R
190
Micro-organism
Water R
pH 7.0
Escherichia coli
NCIB 8666
CIP 2.83
ATCC 9637
C - pH 7.0
35-37 C
pH 8.0
Klebsiella
pneumoniae
NCTC 7427
CIP 53.153
ATCC 10031
C - pH 7.0
35-37 C
Klebsiella
pneumoniae
NCTC 7427
CIP 53.153
ATCC 10031
D - pH 7.0
35-37 C
C - pH 7.0
35-37 C
Erythromycin
ethylsuccinate
Staphylococcus
aureus
NCTC 7447
CIP 53.156
ATCC 6538 P
Framycetin sulphate
Staphylococcus
aureus
NCTC 7447
CIP 53.156
ATCC 6538 P
C - pH 7.0
35-37 C
Antibiotic
Reference
substance
Colistimethate
sodium
Colistimethate
sodium CRS
Dihydrostreptomycin sulphate
Water R
Erythromycin
estolate
Erythromycin CRS
Framycetin
sulphate CRS
Methanol R (see
the monographs)
Water R
pH 8.0
pH 8.0
191
Antibiotic
Gentamicin
sulphate
Reference
substance
Gentamicin
sulphate CRS
Gramicidin CRS
Solvent to be used
Buffer solution
in preparing the
(pH)
stock solution
Micro-organism
Water R
pH 7.0
Staphylococcus
aureus
NCTC 7447
CIP 53.156
ATCC 6538 P
C - pH 7.0
35-37 C
pH 7.0*
Enterococcus hirae
CIP 58.55
ATCC 10541
Staphylococcus
aureus
ATCC 6538 P
C - pH 7.0
35-37 C
Methanol R
Gramicidin
*
Addition of a detergent may be necessary to avoid adsorption on the material during the dilutions, for example 0.1 mg/ml
of polysorbate 80 R
Josamycin
Josamycin
propionate
Kanamycin
monosulphate
Kanamycin acid
sulphate
Josamycin CRS
Josamycin
propionate CRS
Methanol R (see
the monograph)
Methanol R (see
the monograph)
Kanamycin
Water R
monosulphate CRS
Neomycin sulphate
Neomycin sulphate for microbiological Water R
assay CRS
Rifamycin sodium
Spiramycin
Streptomycin
sulphate
Rifamycin
sodium CRS
Spiramycin CRS
Methanol R
Methanol R
pH 5.6
Staphylococcus
aureus
CIP 53.156
ATCC 6538 P
NCTC 7447
C - pH 8.0
35-37 C
pH 5.6
Staphylococcus
aureus
CIP 53.156
ATCC 6538 P
NCTC 7447
C - pH 8.0
35-37 C
pH 8.0
Staphylococcus
aureus
NCTC 7447
CIP 53.156
ATCC 6538 P
C - pH 7.0
35-37 C
pH 8.0
Staphylococcus
aureus
NCTC 7447
CIP 53.156
ATCC 6538 P
C - pH 7.0
35-37 C
pH 7.0
Escherichia coli
NCIB 8879
CIP 54.127
ATCC 10536
C - pH 7.0
35-37 C
pH 7.0
Staphylococcus
aureus
NCTC 7447
CIP 53.156
ATCC 6538 P
C - pH 7.0
35-37 C
pH 8.0
Klebsiella
pneumoniae
NCTC 7427
CIP 53.153
ATCC 10031
C - pH 7.0
35-37 C
C - pH 7.0
37 C
Streptomycin
sulphate CRS
Water R
Tylosin CRS
pH 7.0
Staphylococcus
aureus
NCTC 6571
ATCC 9144
CIP 53.154
Tyrothricin
Gramicidin CRS
Alcohol R
Alcohol R
Enterococcus hirae
ATCC 10541
C - pH 7.0
37 C
Vancomycin
hydrochloride
Vancomycin
hydrochloride CRS
pH 8.0
Staphylococcus
aureus
CIP 53.156
ATCC 6538 P
C - pH 7.0
37-39 C
Tylosin for
veterinary use
Tylosin tartrate for
veterinary use
Water R
192
6g
4g
Beef extract
1.5 g
Yeast extract
3g
Glucose monohydrate
1g
15 g
Agar
1000 ml
Water to produce
Medium B
Pancreatic digest of casein
17 g
3g
5g
Sodium chloride
Dipotassium hydrogen phosphate
2.5 g
Glucose monohydrate
2.5 g
Agar
15 g
Polysorbate 80
10 g
Table 2.7.2.-3.
3g
Yeast extract
3.5 g
Sodium chloride
pH
5.8
3.72
Glucose monohydrate
6.0
5.70
3.68 g
6.2
8.60
1.32 g
6.4
12.60
Water to produce
6.6
17.80
6.8
23.65
7.0
29.63
7.2
35.00
7.4
39.50
7.6
42.80
7.8
45.20
8.0
46.80
1g
1000 ml
Medium D
Heart extract
1.5 g
Yeast extract
1.5 g
Peptone-casein
5g
Glucose monohydrate
1g
Sodium chloride
3.5 g
3.68 g
1.32 g
Potassium nitrate
2g
Water to produce
1000 ml
193
Medium E
Peptone
5g
Meat extract
3g
26.9 g
10 g
Agar
1000 ml
Water to produce
9.4 g
Yeast extract
4.7 g
Beef extract
2.4 g
Sodium chloride
10.0 g
Glucose monohydrate
10.0 g
Agar
23.5 g
Water to produce
1000 ml
REAGENTS
Glycerol
10 g
194